- Email: [email protected]
Hyperexciting developments with voltage-gated Na+channels!
Research Update
2 Richards, D.A. et al. (2000) Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron 27, 551–559 3 Heuser, J.E. and Reese, T.S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344 4 Ceccarelli, B. et al. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57, 499–524 5 Miller, T.M. and Heuser, J.E. (1984) Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction. J. Cell Biol. 98, 685–698 6 Takei, K. et al. (1996) The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin. J. Cell Biol. 133, 1237–1250 7 Fesce, R. et al. (1994) Neurotransmitter release: fusion or ‘kiss and run’? Trends Cell Biol. 4, 1–4 8 Roos, J. and Kelly, R.B. (1999) The endocytic machinery in nerve terminals surrounds sites of exocytosis. Curr. Biol. 9, 1411–1414 9 Stevens, C.F. and Williams, J.H. (2000) ‘Kiss and run’ exocytosis at hippocampal synapses. Proc. Natl. Acad. Sci. U. S. A. 97, 12828–12833 10 Pyle, J.L. et al. (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28, 221–231
TRENDS in Neurosciences Vol.24 No.4 April 2001
11 Südhof, T.C. (2000) The synaptic vesicle cycle revisited. Neuron 28, 317–320 12 von Gersdorff, H. and Matthews, G. (1994) Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367, 735–739 13 von Gersdorff, H. and Matthews, G. (1997) Depletion and replenishment of vesicle pools at a ribbon-type synaptic terminal. J. Neurosci. 17, 1919–1927 14 Koenig, J.H. and Ikeda, K. (1996) Synaptic vesicles have two distinct recycling pathways. J. Cell Biol. 135, 797–808 15 Koenig, J.H. and Ikeda, K. (1999) Contribution of active zone subpopulation of vesicles to evoked and spontaneous release. J. Neurophysiol. 81, 1495–1505 16 Teng, H. et al. (1999) Endocytic active zones: hot spots for endocytosis in vertebrate neuromuscular terminals. J. Neurosci. 19, 4855–4866 17 Teng, H. and Wilkinson, R.S. (2000) Clathrinmediated endocytosis near active zones in snake motor boutons. J. Neurosci. 20, 7986–7993 18 Kuromi, H. and Kidokoro, Y. (1998) Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire. Neuron 20, 917–925
197
19 Kuromi, H. and Kidokoro, Y. (1999) The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae. J. Neurosci. 19, 1557–1565 20 Betz, W.J. et al. (1992) Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12, 363–375 21 Betz, W.J. et al. (1996) Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6, 365–371 22 Ryan, T.A. et al. (1996) The timing of synaptic vesicle endocytosis. Proc. Natl. Acad. Sci. U. S. A. 93, 5567–5571 23 Klingauf, J. et al. (1998) Kinetics and regulation of fast endocytosis at hippocampal synapses. Nature 394, 581–585
Robert S. Wilkinson* John C. Cole Dept of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA. *e-mail: [email protected]
Meeting Report
Hyperexciting developments with voltage-gated Na+ channels! John N. Wood Sodium Channels and Neuronal Hyperexcitability. Held at the Novartis Foundation, London, UK. 14–16, November, 2000.
All the genes encoding functional mammalian voltage-gated Na+ channels might now have been discovered. The tools provided by molecular genetics have provided insights into how channels work, and have allowed the functional significance of Na+ channels in the pathogenesis of epilepsy, pain and muscle dysfunction to be determined. The functional diversity of channels, their patterns of expression, their interactions with regulatory subunits, and association of subtle Na+ channel mutations with disease states were recently reviewed. A recent meeting held at the Novartis Foundation in London, organized and chaired by Steve Waxman (Yale University, USA) brought together members of the Na+ channel community. The presentations and discussion will be published in Sodium Channels and Neuronal Hyperexcitability, Vol. 241 in
the Novartis Foundation series in 2001. Voltage-gated Na+ channels comprise a family of proteins whose conservation of structure is belied by a bewildering nomenclature, which has recently been streamlined1. The functional α-subunits, which contain the channel pore-forming regions and voltage sensors, all map within four paralogous chromosome segments adjacent to the HOX gene clusters. Within each group, duplications have given rise to a family of ten channel genes in mammals2. The functional channels are single large proteins that comprise four repeated domains of six transmembrane segments folded into Na+-selective pores that are activated by changes in membrane potential3. Two of the channels are primarily expressed in muscle, whereas the others are expressed in complex patterns in central and peripheral neurons and glia. The structural features associated with channel gating and inactivation were discussed in detail by Catterall (Washington, USA)and Horn (Philadelphia, USA) and correlated with
electrophysiological studies by Keynes (Cambridge, UK). The voltage sensor role of the positively charged residues in the four S-4 domains of voltage-gated channels is now well established experimentally using cysteine-scanning mutagenesis (Horn). These segments sit in water-filled crevices and might move in a screw-helical fashion very rapidly (milliseconds) through a gating pore in response to changes in membrane potential. The channel pore itself is formed by the S5 and S6 segments linked through a short extracellular loop. Fast inactivation involves the intracellular loop between domains III and IV, the structure of which has recently been determined by NMR. A hydrophobic tripeptide, IFM, plays a crucial role in fast inactivation. The sites of binding of many Na+ channel blockers, including local anaesthetics, are located in S6 segments of domains 3 and 4. Scorpion peptide toxins that alter channel gating bind to S4 voltage sensors and lock the channels in activated or non-inactivated states.
http://tins.trends.com 0166-2236/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01762-8
198
Research Update
Now that the human and mouse genome sequencing exercises are almost complete it seems unlikely that we will find any more functional α-subunits. By contrast, additional accessory subunits that regulate Na+ channel function and localization will probably be identifed, as the recent identification of two novel βsubunits – β-1A and β-3 – has shown. Indeed, the demonstration of interactions of Na+ channels with many proteins, including both extracellular and cytoskeletal elements, in addition to tyrosine phosphatases and synaptotagmin suggest that other accessory subunits could exist (Isom, Ann Arbor, USA and Catterall). Knockout studies of β-subunits are likely to be very illuminating in understanding the physiological significance of βsubunits, which have already been demonstrated to regulate channel expression and kinetics, in addition to playing a role in their sub-cellular localization (Isom). What are all these Na+ channels for? Three routes to understanding the specialized role of Na+ channels are available. First, and most obviously, the expression pattern of Na+ channels in different neuronal subsets provides clues to their function. Second, the expression and analysis of Na+ channels in vitro clearly shows differences in pharmacology and kinetics of the channels that probably underlie different physiological roles. Finally, association of various pathological states with particular dysfunctional Na+ channels is indicative of the normal physiological role of channel isoforms. Hyperexcitability, as exemplified by epileptic activity, might be intimately associated with Na+ channel dysregulation. The persistent Na+
TRENDS in Neurosciences Vol.24 No.4 April 2001
channel activity that can result in epileptiform activity was discussed by Segal (Harvard, USA) using micro-island cultures of hippocampal neurons. This system provides an interesting route for the identification of anticonvulsants that might act through suppression of the persistent Na+ channel activity that occurs at depolarized voltages. The dramatic effects of Na+ channel dysregulation that result in some forms of epilepsy are beginning to be understood, and other more subtle disorders might also involve Na+ channels. Ptacek (HHMI, Utah, USA) discussed mutants of Nav1.4 that give rise to familial periodic paralyses characterized by episodes of muscle weakness that are induced by stress or fatigue. Mutations in Nav1.6 also account for the muscle weakness in the motor end plate (med) mouse mutant, in addition to the changes in some cerebellar neurons. A modifier locus that results in perinatal lethality in the med mouse has also been identified by Meisler (Ann Arbor, USA), the precise sequence for which should soon be known. A variety of channelopathies involve either mutant α or β subunits. Meisler has found mutations in Nav1.1 that result in generalized epilepsy with febrile seizures type II, whereas mutations in the β-1 subunit cause the type I syndrome. The recent discovery that the cardiac channel Nav1.5 is also expressed in the limbic system might explain why mutations in this channel Nav1.5 lead not only to LQT syndrome but also to epilepsy (Noebels, Baylor, USA). Indirect effects on channel expression can also have deleterious effects. In various demyelinating diseases, aberrant Na+ channel expression occurs within the CNS [for example Nav1.8 is abnormally expressed in Purkinje cells (Waxman)].
Interestingly, mis-expression of Na+ channels as a result of demyelinating diseases is associated with the seizure activity seen in myelin basic protein (MBP)−/− mice, which show high levels of expression of Nav1.2 in demyelinated neurons (Noebels). Waxman overviewed the pattern and plasticity of expression of Na+ channel genes in sensory neurons in various pain pathologies, and correlated altered channel expression with changes in excitability, a subject also briefly addressed by Bevan (Novartis, UK) and Wood (London, UK) who emphasized why there is increasing interest in Na+ channels as potential analgesic drug targets, particularly in the context of neuropathic pain. This brief meeting underscored how far we have come in the past decade in appreciating the complexity of different types of Na+ channels in the function of excitable tissues. Identification of new mutant, polymorphic channels or their regulatory genes might well identify therapeutic targets for pathologies, other than those discussed at the meeting. These are indeed exciting times in Na+ channel studies. References 1 Goldin, A.L. et al. (2000) Nomenclature of voltage-gated sodium channels. Neuron 28, 365–368 2 Plummer, N.W. and Meisler, M.H. (1999) Evolution and diversity of mammalian sodium channel genes. Genomics 15, 323–331 3 Catterall, W.A. (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26, 13–25
John N. Wood Biology Dept, University College London, Gower Street, London, UK WC1E 6BT. e-mail: [email protected]
What does the human genome sequence mean to you? For a thorough and independent analysis of the meaning and importance of the February publications of the draft human genome sequences, visit http://news.bmn.com/hmsbeagle/96/notes/feature3. To mark the importance of the event, we are pre-publishing a number of Trends articles on these publications in our free, online magazine, HMS Beagle. You cannot afford to miss these up-to-the-minute commentaries, opinions and updates, written by leading players from across the whole of biology. And they're free!
http://tins.trends.com
2 Richards, D.A. et al. (2000) Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron 27, 551–559 3 Heuser, J.E. and Reese, T.S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344 4 Ceccarelli, B. et al. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57, 499–524 5 Miller, T.M. and Heuser, J.E. (1984) Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction. J. Cell Biol. 98, 685–698 6 Takei, K. et al. (1996) The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin. J. Cell Biol. 133, 1237–1250 7 Fesce, R. et al. (1994) Neurotransmitter release: fusion or ‘kiss and run’? Trends Cell Biol. 4, 1–4 8 Roos, J. and Kelly, R.B. (1999) The endocytic machinery in nerve terminals surrounds sites of exocytosis. Curr. Biol. 9, 1411–1414 9 Stevens, C.F. and Williams, J.H. (2000) ‘Kiss and run’ exocytosis at hippocampal synapses. Proc. Natl. Acad. Sci. U. S. A. 97, 12828–12833 10 Pyle, J.L. et al. (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28, 221–231
TRENDS in Neurosciences Vol.24 No.4 April 2001
11 Südhof, T.C. (2000) The synaptic vesicle cycle revisited. Neuron 28, 317–320 12 von Gersdorff, H. and Matthews, G. (1994) Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367, 735–739 13 von Gersdorff, H. and Matthews, G. (1997) Depletion and replenishment of vesicle pools at a ribbon-type synaptic terminal. J. Neurosci. 17, 1919–1927 14 Koenig, J.H. and Ikeda, K. (1996) Synaptic vesicles have two distinct recycling pathways. J. Cell Biol. 135, 797–808 15 Koenig, J.H. and Ikeda, K. (1999) Contribution of active zone subpopulation of vesicles to evoked and spontaneous release. J. Neurophysiol. 81, 1495–1505 16 Teng, H. et al. (1999) Endocytic active zones: hot spots for endocytosis in vertebrate neuromuscular terminals. J. Neurosci. 19, 4855–4866 17 Teng, H. and Wilkinson, R.S. (2000) Clathrinmediated endocytosis near active zones in snake motor boutons. J. Neurosci. 20, 7986–7993 18 Kuromi, H. and Kidokoro, Y. (1998) Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire. Neuron 20, 917–925
197
19 Kuromi, H. and Kidokoro, Y. (1999) The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae. J. Neurosci. 19, 1557–1565 20 Betz, W.J. et al. (1992) Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12, 363–375 21 Betz, W.J. et al. (1996) Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6, 365–371 22 Ryan, T.A. et al. (1996) The timing of synaptic vesicle endocytosis. Proc. Natl. Acad. Sci. U. S. A. 93, 5567–5571 23 Klingauf, J. et al. (1998) Kinetics and regulation of fast endocytosis at hippocampal synapses. Nature 394, 581–585
Robert S. Wilkinson* John C. Cole Dept of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA. *e-mail: [email protected]
Meeting Report
Hyperexciting developments with voltage-gated Na+ channels! John N. Wood Sodium Channels and Neuronal Hyperexcitability. Held at the Novartis Foundation, London, UK. 14–16, November, 2000.
All the genes encoding functional mammalian voltage-gated Na+ channels might now have been discovered. The tools provided by molecular genetics have provided insights into how channels work, and have allowed the functional significance of Na+ channels in the pathogenesis of epilepsy, pain and muscle dysfunction to be determined. The functional diversity of channels, their patterns of expression, their interactions with regulatory subunits, and association of subtle Na+ channel mutations with disease states were recently reviewed. A recent meeting held at the Novartis Foundation in London, organized and chaired by Steve Waxman (Yale University, USA) brought together members of the Na+ channel community. The presentations and discussion will be published in Sodium Channels and Neuronal Hyperexcitability, Vol. 241 in
the Novartis Foundation series in 2001. Voltage-gated Na+ channels comprise a family of proteins whose conservation of structure is belied by a bewildering nomenclature, which has recently been streamlined1. The functional α-subunits, which contain the channel pore-forming regions and voltage sensors, all map within four paralogous chromosome segments adjacent to the HOX gene clusters. Within each group, duplications have given rise to a family of ten channel genes in mammals2. The functional channels are single large proteins that comprise four repeated domains of six transmembrane segments folded into Na+-selective pores that are activated by changes in membrane potential3. Two of the channels are primarily expressed in muscle, whereas the others are expressed in complex patterns in central and peripheral neurons and glia. The structural features associated with channel gating and inactivation were discussed in detail by Catterall (Washington, USA)and Horn (Philadelphia, USA) and correlated with
electrophysiological studies by Keynes (Cambridge, UK). The voltage sensor role of the positively charged residues in the four S-4 domains of voltage-gated channels is now well established experimentally using cysteine-scanning mutagenesis (Horn). These segments sit in water-filled crevices and might move in a screw-helical fashion very rapidly (milliseconds) through a gating pore in response to changes in membrane potential. The channel pore itself is formed by the S5 and S6 segments linked through a short extracellular loop. Fast inactivation involves the intracellular loop between domains III and IV, the structure of which has recently been determined by NMR. A hydrophobic tripeptide, IFM, plays a crucial role in fast inactivation. The sites of binding of many Na+ channel blockers, including local anaesthetics, are located in S6 segments of domains 3 and 4. Scorpion peptide toxins that alter channel gating bind to S4 voltage sensors and lock the channels in activated or non-inactivated states.
http://tins.trends.com 0166-2236/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01762-8
198
Research Update
Now that the human and mouse genome sequencing exercises are almost complete it seems unlikely that we will find any more functional α-subunits. By contrast, additional accessory subunits that regulate Na+ channel function and localization will probably be identifed, as the recent identification of two novel βsubunits – β-1A and β-3 – has shown. Indeed, the demonstration of interactions of Na+ channels with many proteins, including both extracellular and cytoskeletal elements, in addition to tyrosine phosphatases and synaptotagmin suggest that other accessory subunits could exist (Isom, Ann Arbor, USA and Catterall). Knockout studies of β-subunits are likely to be very illuminating in understanding the physiological significance of βsubunits, which have already been demonstrated to regulate channel expression and kinetics, in addition to playing a role in their sub-cellular localization (Isom). What are all these Na+ channels for? Three routes to understanding the specialized role of Na+ channels are available. First, and most obviously, the expression pattern of Na+ channels in different neuronal subsets provides clues to their function. Second, the expression and analysis of Na+ channels in vitro clearly shows differences in pharmacology and kinetics of the channels that probably underlie different physiological roles. Finally, association of various pathological states with particular dysfunctional Na+ channels is indicative of the normal physiological role of channel isoforms. Hyperexcitability, as exemplified by epileptic activity, might be intimately associated with Na+ channel dysregulation. The persistent Na+
TRENDS in Neurosciences Vol.24 No.4 April 2001
channel activity that can result in epileptiform activity was discussed by Segal (Harvard, USA) using micro-island cultures of hippocampal neurons. This system provides an interesting route for the identification of anticonvulsants that might act through suppression of the persistent Na+ channel activity that occurs at depolarized voltages. The dramatic effects of Na+ channel dysregulation that result in some forms of epilepsy are beginning to be understood, and other more subtle disorders might also involve Na+ channels. Ptacek (HHMI, Utah, USA) discussed mutants of Nav1.4 that give rise to familial periodic paralyses characterized by episodes of muscle weakness that are induced by stress or fatigue. Mutations in Nav1.6 also account for the muscle weakness in the motor end plate (med) mouse mutant, in addition to the changes in some cerebellar neurons. A modifier locus that results in perinatal lethality in the med mouse has also been identified by Meisler (Ann Arbor, USA), the precise sequence for which should soon be known. A variety of channelopathies involve either mutant α or β subunits. Meisler has found mutations in Nav1.1 that result in generalized epilepsy with febrile seizures type II, whereas mutations in the β-1 subunit cause the type I syndrome. The recent discovery that the cardiac channel Nav1.5 is also expressed in the limbic system might explain why mutations in this channel Nav1.5 lead not only to LQT syndrome but also to epilepsy (Noebels, Baylor, USA). Indirect effects on channel expression can also have deleterious effects. In various demyelinating diseases, aberrant Na+ channel expression occurs within the CNS [for example Nav1.8 is abnormally expressed in Purkinje cells (Waxman)].
Interestingly, mis-expression of Na+ channels as a result of demyelinating diseases is associated with the seizure activity seen in myelin basic protein (MBP)−/− mice, which show high levels of expression of Nav1.2 in demyelinated neurons (Noebels). Waxman overviewed the pattern and plasticity of expression of Na+ channel genes in sensory neurons in various pain pathologies, and correlated altered channel expression with changes in excitability, a subject also briefly addressed by Bevan (Novartis, UK) and Wood (London, UK) who emphasized why there is increasing interest in Na+ channels as potential analgesic drug targets, particularly in the context of neuropathic pain. This brief meeting underscored how far we have come in the past decade in appreciating the complexity of different types of Na+ channels in the function of excitable tissues. Identification of new mutant, polymorphic channels or their regulatory genes might well identify therapeutic targets for pathologies, other than those discussed at the meeting. These are indeed exciting times in Na+ channel studies. References 1 Goldin, A.L. et al. (2000) Nomenclature of voltage-gated sodium channels. Neuron 28, 365–368 2 Plummer, N.W. and Meisler, M.H. (1999) Evolution and diversity of mammalian sodium channel genes. Genomics 15, 323–331 3 Catterall, W.A. (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26, 13–25
John N. Wood Biology Dept, University College London, Gower Street, London, UK WC1E 6BT. e-mail: [email protected]
What does the human genome sequence mean to you? For a thorough and independent analysis of the meaning and importance of the February publications of the draft human genome sequences, visit http://news.bmn.com/hmsbeagle/96/notes/feature3. To mark the importance of the event, we are pre-publishing a number of Trends articles on these publications in our free, online magazine, HMS Beagle. You cannot afford to miss these up-to-the-minute commentaries, opinions and updates, written by leading players from across the whole of biology. And they're free!
http://tins.trends.com